Rapid processing of tissues for histology offers important advantages for rapid diagnosis, convenience and efficiency. The earliest methods for decreasing processing times involved heating specimens [1, 2]. Introduction of the microwave processor into the laboratory offered a more controlled approach to heating tissues and cells. By penetrating the biological material, microwaves overcame the limitation of poor heat conduction and thus produced more even heating throughout the processed specimens. Introduced in 1970 for histological processing [3] microwave fixation produced satisfactory results with few artifacts in mouse and fresh human postmortem tissues [3]. The idea of heating tissues in a constant volume bath of liquid to stabilize the temperatures inside the microwave chamber was introduced in 1978 [4] when Login performed a study to compare the effects of heating tissues in different solutions. Heating tissues in saline or Zenker’s solution (a fixative used by histologists that contains mercuric chloride and acetic acid) produced better results than if tissues were heated in formalin [4]. Enzyme reactivity and immunoreactivity was preserved in microwave-fixed tissues [5], microwave-assisted heating of sections was discovered to be an efficient method for antigen retrieval [6], and immunolabeling times could be shortened using microwaves [7–9]. Microwave irradiation was also found to decrease decalcification rates of human temporal bones with no detectable adverse effects on ultrastructure or antigenicity [10–13]. The microwave processor thus became a routine tool in the histology laboratory for heating biological specimens and speeding up processing times [14–18].

Introduction of microwave technologies to the electron microscopy laboratory were initially restricted to methods involving specimen heating [5, 19]. Although the microwave-induced heating produced useable preserved specimens, the ultrastructural damage was not suitable for critical work [5, 19, 20]. Microwave-induced heating in the presence of aldehyde gradually took over as a viable approach to rapid fixation for electron microscopy [20–23]. Microwave processing under more careful temperature control was achieved using water loads in the chamber of the processor, or by shielding the tissues with cooling jackets containing circulating water [24, 25]. Careful evaluation of microwave-assisted processing of biological material began to reveal improved morphology when compared with conventional processing methods performed at ambient temperature [26–30]. Examination of microwave-processed bone revealed improved ultrastructure and antigen retention [12, 31].

The technical developments that allowed biological material to be exposed to microwaves without being heated [29], opened a controversy that is still not completely settled, that of whether there is a “microwave-only effect” independent of temperature. Early studies suggested that specimen heating caused by exposure to microwaves was responsible for the decrease in preparation times and improved ultrastructure [20, 32]. However, more recent studies using more carefully controlled conditions seem to indicate a role for microwaves in the absence of heat in the improvements of processing times and ultrastructure [29, 33]. Electromagnetic effects of microwaves on cellular membranes, supporting a microwave-only effect, have been demonstrated in Escherichia coli bacteria [34].

Microwave processors have become essential tools in the histology laboratory and have been incorporated into routine protocols. Microwaves are used to assist in specimen fixation [16, 35, 36], paraffin embedding [37, 38], antigen retrieval [6, 39, 40], staining [15, 41, 42], immunolabeling [7, 9, 39, 43–47], and in situ hybridization [48, 49]. Microwave processors are also being used for rapid bone decalcification [10–13, 33] (see Fig. 1).

Similar reports of microwave-assisted processing for electron microscopy indicate rapid processing times, improved specimen morphology and increased antigenicity of specimens being used for immunocytochemical experiments [21, 26, 28, 29, 31, 47, 50–54]. Microwave assisted resin polymerization has been used to embed thawed, thin cryosections [55], and diluted antibodies have been microwave irradiated to increase their labeling efficiency for EM immunocytochemistry [46]. As a result of these reports, and with the inclusion of microwave processing in the curricula of practical courses, electron microscopy laboratories are adapting the microwave processor for use in routine protocols.

The protocols described in this chapter have been extensively tested in different laboratories using different microwave processors and have been shown to yield reproducible results. The only variable we have discovered is with the Epon substitute available from different suppliers. The Spurr-Epon recipe we have provided will polymerize differently if Eponate 12 is not used. However, it appears that all Epon substitutes can be used, but the final result may require some adjustment to optimize the hardness of the resin. We recommend using complete kits from a single supplier, and not mixing ingredients from different suppliers. We also recommend testing all resin mixtures before using them to embed specimens.

The complete protocol can be applied as is, or, as is the case in my laboratory, parts of the protocol can be incorporated into existing long protocols. For example, we occasionally perform dehydration and resin infiltration using the microwave processor but perform the final resin polymerization at 60 °C in a regular oven. We also use rapid resin polymerization to re-embed polymerized specimens that need reorienting for sectioning. Examples of microwave assisted processing can be examined in Figs. 2, 3, and 4 in this chapter. All were processed into epoxy resin using microwave-assisted fixation, dehydration, and resin infiltration using the protocol outlined below and annotated in Table 1(a). All specimens were infiltrated using the epoxy resin formulation documented in Table 2(c). The specimens illustrated in Fig. 2 were polymerized in resin using the microwave protocol described in Subheading 3.6. Specimens illustrated in Figs. 3 and 4 were polymerized in resin using an overnight exposure to 60 °C.